Immunization of dairy cattle with GapC protein against Streptococcus infection

ABSTRACT

The GapC plasmin binding protein genes of  Streptococcus dysgalactiae  ( S. dysgalactiae ),  Streptococcus agalactiae  ( S. agalactiae ),  Streptococcus uberis  ( S. uberis ),  Streptococcus parauberis  ( S. parauberis ), and  Streptococcus iniae  ( S. iniae ) are described, as well as the recombinant production of the GapC proteins therefrom. Also described is the use of the GapC proteins from those species in vaccine compositions to prevent or treat bacterial infections in general, and mastitis in particular.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to provisional patent application Ser. No.60/211,022, filed Jun. 12, 2000, from which application priority isclaimed under 35 USC § 119(e)(1) and which application is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present invention relates generally to bacterial antigens and genesencoding the same. More particularly, the present invention pertains tothe cloning, expression and characterization of the GapC plasmin-bindingproteins from Streptococcus dysgalactiae, Streptococcus agalactiae,Streptococcus uberis, Streptococcus parauberis, and Streptococcus iniae,and the use of the same in vaccine compositions.

BACKGROUND

Mastitis is an infection of the mammary gland usually caused by bacteriaor fungus. The inflammatory response following infection results indecreased milk yield as well as quality, and causes major annualeconomic losses to the dairy industry.

Among the bacterial species most commonly associated with mastitis arevarious species of the genus Streptococcus, including Streptococcusaureus, Streptococcus uberis (untypeable), Streptococcus agalactiae(Lancefield group B), Streptococcus dysgalactiae (Lancefield group C),Streptococcus zooepidemicus, and the Lancefield groups D, G, L and Nstreptococci. Some of those species are contagions (e.g. S. agalactiae),while others are considered environmental pathogens (e.g. S.dysgalactiae and S. uberis).

The environmental pathogen S. uberis is responsible for about 20% of allclinical cases of mastitis (Bramley, A. J. and Dodd, F. H. (1984) J.Dairy Res. 51:481-512; Bramley, A. J. (1987) Animal Health Nutrition42:12-16; Watts, J. L. (1988) J. Dairy Sci. 71:1616-1624); it is thepredominant organism isolated from mammary glands during thenon-lactating period (Bramley, A. J. (1984) Br. Vet. J. 140:328-335;Bramley and Dodd (1984) J. Dairy Res. 51:481-512; Oliver, S. P. (1988)Am. J. Vet. Res. 49:1789-1793).

Mastitis resulting from infection with S. uberis is commonlysubclinical, characterized by apparently normal milk with an increase insomatic cell counts due to the influx of leukocytes. The chemicalcomposition of milk is changed due to suppression of secretion with thetransfer of sodium chloride and bicarbonate from blood to milk, causinga shift of pH to a more alkaline level. S. uberis mastitis may also takethe form of an acute clinical condition, with obvious signs of diseasesuch as clots or discoloration of the milk and swelling or hardness ofthe mammary gland. Some cases of the clinical disease can be severe andpyrexia may be present. For a review of the clinical manifestations ofS. uberis mastitis, see, Bramley (1991) Mastitis: physiology orpathology. p. 3-9. In C. Burvenich, G. Vandeputte-van Messom, and A. W.Hill (ed.), New insights into the pathogenesis of mastitis.Rijksuniversiteit Gent, Belgium; and Schalm et al. (1971) The mastitiscomplex-A brief summary. p. 1-3. In Bovine Mastitis. Lea & Febiger,Philadelphia

Conventional antibacterial control methods such as teat dipping andantibiotic therapy are effective in the control of many types ofcontagious mastitis, but the environmental organisms typically found inall dairy barns are often resistant to such measures. Vaccination istherefore an attractive strategy to prevent infections of the mammaryglands, and has been shown to be beneficial in the case of somecontagious mastitis pathogens.

The literature is limited regarding vaccination studies with S.dysgalactiae and S. uberis, and variable results have been observed. Insome cases, immunization has resulted in increased sensitivity to thespecific organism and in other cases strain-specific protection has beenobtained.

For example, previous studies have shown that primary infection with S.uberis can considerably reduce the rate of infection following a secondchallenge with the same strain (Hill, A. W. (1988) Res. Vet. Sci.44:386-387). Local vaccination with killed S. uberis protects the bovinemammary gland against intramammary challenge with the homologous strain(Finch et al. (1994) Infect. Immun. 62:3599-3603). Similarly,subcutaneous vaccination with live S. uberis has been shown to cause adramatic modification of the pathogenesis of mastitis with the samestrain (Hill et al. (1994) FEMS Immunol. Med. Microbiol. 8:109-118).Animals vaccinated in this way shed fewer bacteria in their milk andmany quarters remain free of infection.

Nonetheless, vaccination with live or attenuated bacteria can pose risksto the recipient. Further, it is clear that conventional killed vaccinesare in general largely ineffective against S. uberis and S. agalactiae,either due to lack of protective antigens on in vitro-grown cells ormasking of these antigens by molecular mimicry.

The current lack of existing mastitis vaccines against S. agalactiae orthe contagious streptococcus strains is due at least in part to a lackof knowledge regarding the virulence determinants and protectiveantigens produced by those organisms which are involved in invasion andprotection of the mammary gland (Collins et al. (1988) J. Dairy Res.55:25-32; Leigh et al. (1990) Res. Vet. Sci. 49: 85-87; Marshall et al.(1986) J. Dairy Res. 53: 507-514).

S. dysgalactiae is known to bind several extracellular andplasma-derived proteins such as fibronectin, fibrinogen, collagen,alpha-II-macroglobulin, IgG, albumin and other compounds. The organismalso produces hyaluronidase and fibrinolysin and is capable of adheringto and invading bovine mammary epithelial cells. However, the exactroles of the bacterial components responsible for these phenotypes inpathogenesis is not known.

Similarly, the pathogenesis of S. uberis infection is poorly understood.Furthermore, the influence of S. uberis virulence factors on hostdefense mechanisms and mammary gland physiology is not well defined.Known virulence factors associated with S. uberis include a hyaluronicacid capsule (Hill, A. W. (1988) Res. Vet. Sci. 45:400-404),hyaluronidase (Schaufuss et al. (1989) Zentralbl. Bakteriol. Ser. A271:46-53), R-like protein (Groschup, M. H. and Timoney, J. F. (1993)Res. Vet. Sci. 54:124-126), and a cohemolysin, the CAMP factor, alsoknown as UBERIS factor (Skalka, B. and Smola, J. (1981) Zentralbl.Bakteriol. Ser. A 249:190-194), R-like protein, plasminogen activatorand CAMP factor. However, very little is known of their roles inpathogenicity.

The use of virulence determinants from Streptococcus as immunogenicagents has been proposed. For example, the CAMP factor of S. uberis hasbeen shown to protect vertebrate subjects from infection by thatorganism (Jiang, U.S. Pat. No. 5,863,543).

The γ antigen of the group B Streptococci strain A909 (ATCC No. 27591)is a component of the c protein marker complex, which additionallycomprises an α and β subunit (Boyle, U.S. Pat. No. 5,721,339). Subsetsof serotype Ia, II, and virtually all serotype Ib cells of group Bstreptococci, have been reported to express components of the c protein.Use of the γ subunit as an immunogenic agent against infections byLancefield Group B Streptococcus infection has been proposed. However,its use to prevent or treat bacterial infections in animals, includingmastitis in cattle, has not been studied.

The group A streptococcal M protein is considered to be one of the majorvirulence factors of this organism by virtue of its ability to impedeattack by human phagocytes (Lancefield, R. C. (1962) J. Immunol.89:307-313). The bacteria persist in the infected tissue untilantibodies are produced against the M molecule. Type-specific antibodiesto the M protein are able to reverse the antiphagocytic effect of themolecule and allow efficient clearance of the invading organism.

M proteins are one of the key virulence factors of Streptococcuspyogenes, due to their involvement in mediating resistance tophagocytosis (Kehoe, M. A. (1991) Vaccine 9:797-806) and their abilityto induce potentially harmful host immune responses via theirsuperantigenicity and their capacity to induce host-cross-reactiveantibody responses (Bisno, A. L. (1991) New Engl. J. Med. 325:783-793;Froude et al. (1989) Curr. Top. Microbiol. Immunol. 145:5-26;Stollerman, G. H. (1991) Clin. Immunol. Immunopathol. 61:131-142).

However, obstacles exist to using intact M proteins as vaccines. Theprotein's opsonic epitopes are extremely type-specific, resulting innarrow, type-specific protection. Further, some M proteins appear tocontain epitopes that cross react with tissues of the immunized subject,causing a harmful autoimmune response (See e.g., Dale, J. L. andBeached, G. H. (1982) J. Exp. Med 156:1165-1176; Dale, J. L. andBeached, G. H. (1985) J. Exp. Med. 161:113-122; Baird, R. W., Bronze, M.S., Drabs, W., Hill, H. R., Veasey, L. G. and Dale, J. L. (1991) J.Immun. 146:3132-3137; Bronze, M. S. and Dale, J. L. (1993) J. Immun151:2820-2828; Cunningham, M. W. and Russell, S. M. (1983) Infect.Immun. 42:531-538).

Chimeric proteins containing three different fibronectin binding domains(FNBDs) derived from fibronectin binding proteins of S. dysgalactiae andStaphylococcus aureus have been expressed on the surface of Staph.carnosus cells. In the case of one of these proteins, intranasalimmunizations with live recombinant Staph. carnosus cells expressing thechimeric protein on their surface resulted in an improved antibodyresponse to a model immunogen present within the chimeric surfaceprotein.

A GapC plasmin binding protein from a strain of Group A Streptococcushas previously been identified and characterized, and its use inthrombolytic therapies has been described (Boyle, et al., U.S. Pat. No.5,237,050; Boyle, et al., U.S. Pat. No. 5,328,996).

However, until now, the protective capability of GapC has not beenstudied, nor have the GapC proteins of Streptococcus dysgalactiae,Streptococcus agalactiae, Streptococcus uberis, Streptococcus parauberisor Streptococcus iniae been isolated or characterized.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides novel Streptococcus GapCproteins and vaccine compositions comprising the same. In oneembodiment, the invention is directed to an isolated GapC proteinselected from the group consisting of:

(a) an isolated Streptococcus dysgalactiae GapC protein comprising theamino acid sequence shown at amino acid positions 1 to 336, inclusive,of FIGS. 1A-1B (SEQ ID NO:4);

(b) an isolated Streptococcus agalactiae GapC protein comprising theamino acid sequence shown at amino acid positions 1 to 336, inclusive,of FIGS. 2A-2B (SEQ ID NO:6);

(c) an isolated Streptococcus uberis GapC protein comprising the aminoacid sequence shown at amino acid positions 1 to 336, inclusive, ofFIGS. 3A-3B (SEQ ID NO:8);

(d) an isolated Streptococcus parauberis GapC protein comprising theamino acid sequence shown at amino acid positions 1 to 336, inclusive,of FIGS. 4A-4B (SEQ ID NO:10);

(e) an isolated Streptococcus iniae GapC protein comprising the aminoacid sequence shown at amino acid positions 1 to 336, inclusive, ofFIGS. 5A-5B (SEQ ID NO:12); and

(f) immunogenic fragments of (a), (b), (c), (d) and (e) comprising atleast about 5 amino acids.

In another embodiment, the invention is directed to isolatedpolynucleotides comprising coding sequences for the above-described GapCproteins, or complements thereof, recombinant vectors comprising thepolynucleotides, host cells comprising the recombinant vectors, andmethods of recombinantly producing the GapC proteins.

In yet other embodiments, the subject invention is directed to vaccinecompositions comprising a pharmaceutically acceptable vehicle and a GapCprotein as described above, methods of producing the vaccinecompositions, as well as methods of treating or preventing bacterialinfections in a vertebrate subject comprising administering to thesubject a therapeutically effective amount of the vaccine composition.The bacterial infection is, for example, a streptococcus infection andmay cause mastitis. The vaccine compositions may further comprise anadjuvant.

In other embodiments, the invention is directed to antibodies directedagainst the isolated GapC proteins. The antibodies may be polyclonal ormonoclonal.

In still further embodiments, the invention is directed to methods ofdetecting Streptococcus antibodies in a biological sample, comprising:

(a) reacting the biological sample with an isolated GapC protein underconditions which allow said Streptococcus antibodies, when present inthe biological sample, to bind to the GapC protein to form anantibody/antigen complex; and

(b) detecting the presence or absence of the complex, thereby detectingthe presence or absence of Streptococcus antibodies in said sample.

In another embodiment, the invention is directed to a method ofdetecting a GapC protein in a biological sample, comprising:

(a) reacting the sample with antibodies directed against the GapCprotein under conditions which allow the antibodies to bind to the GapCprotein, when present in the sample, to form an antibody/antigencomplex; and

(b) detecting the presence or absence of the complex, thereby detectingthe presence or absence of a GapC protein in the sample.

In another embodiment, the invention is directed to an immunodiagnostictest kit for detecting Streptococcus infection, the test kit comprisinga GapC protein, or antibodies directed against a GapC protein, andinstructions for conducting the immunodiagnostic test.

These and other embodiments of the subject invention will readily occurto those of skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B depict the isolated nucleotide sequences and deduced aminoacid sequences of the gapC gene for S. dysgalactiae (SEQ ID NO:3 and SEQID NO:4).

FIGS. 2A-2B depict the isolated nucleotide sequences and deduced aminoacid sequences of the gapC gene for S. agalactiae (SEQ ID NO:5 and SEQID NO:6).

FIGS. 3A-3B depict the isolated nucleotide sequences and deduced aminoacid sequences of the gapC gene for S. uberis (SEQ ID NO:7 and SEQ IDNO:8).

FIGS. 4A-4B depict the isolated nucleotide sequences and deduced aminoacid sequences of the gapC gene for S. parauberis (SEQ ID NO:9 and SEQID NO: 10).

FIGS. 5A-5B depict the isolated nucleotide sequences and deduced aminoacid sequences of the gapC gene for S. iniae (SEQ ID NO: 11 and SEQ IDNO: 12).

FIGS. 6A-6E (SEQ ID NOS:3, 5, 7, 9, 11, 13, 15 and 17) show a DNAalignment created by PileUp and displayed by Pretty software (acomponent of the GCG Wisconsin Package, version 10, provided by theSeqWeb sequence analysis package, version 1.1, of the CanadianBioinformatics Resource). The figure depicts the isolated nucleotidesequences of the gapC genes from S. dysgalactiae (DysGapO, Check 9344),S. agalactiae (AgalGapC. Check 2895), S. uberis (UberGapC, Check 5966),S. parauberis (PUberGapC, Check 9672), and S. iniae (IniaeGapC, Check990). The previously known sequences of S. equisimilis (SeqGapC, Check5841 ), S. pyogenes (SpyGapO, Check 4037), and a bovine GAPDH protein(BovGapC, check 5059) are also included. The length and weightparameters were the same for all sequences (1018 and 1.00,respectively). The parameters used in the DNA sequence comparison wereas follows: Plurality—2.00; Threshold—1; AveWeight—1.00; AveMatch—1.00;AvMisMatch—0.00; Symbol comparison table—pileupdna.cmp; CompCheck—6876;Gap Weight—5; GapLengthWeight—1; PileUp MSF—1018; Type—N; Check—3804. Inthe figure, dashes represent identical nucleotides; dots represent gapsintroduced by the software used to generate the alignment chart, andtildes represent regions not included in the overall alignment due todifferences in the length of the gene sequences.

FIGS. 7A-7B (SEQ ID NOS:4, 6, 8, 10, 12, 14, 16 and 18) show an aminoacid sequence alignment created by PileUp and displayed by Pretty (asabove) that depicts the deduced amino acid sequences of the GapCproteins from S. dysgalactiae (DysGapC, Check 6731), S. agalactiae(AgalGapC, Check 1229), S. uberis (UberGapC, Check 8229), S. parauberis(PUberGapC, Check 8889) and S. iniae (IniaeGapC, check 8785). Thepreviously known sequences of S. equisimilis (SeqGapC, Check 8252), S.pyogenes (SpyGapC, Check 6626) and a bovine GAPDH protein (BovGapC,Check 8479) are also included. In the figure, dashes represent identicalamino acid residues; dots represent gaps introduced by the PileUpsoftware, and tildes represent regions not included in the overallalignment due to differences in the length of the gene sequences.

FIG. 8 shows Kyte-Doolittle hydropathy plots (averaged over a window of7), Emini surface probability plots, Karplus-Schulz chain flexibilityplots, Jameson-Wolf antigenic index plots, and both Chou-Fasman andGarnier-Osguthorpe-Robson secondary structure plots for the GapC proteinisolated from S. dysgalactiae.

FIG. 9 shows Kyte-Doolittle hydropathy plots (averaged over a window of7), Emini surface probability plots, Karplus-Schulz chain flexibilityplots, Jameson-Wolf antigenic index plots, and both Chou-Fasman andGamier-Osguthorpe-Robson secondary structure plots for the GapC proteinisolated from S. agalactiae.

FIG. 10 shows Kyte-Doolittle hydropathy plots (averaged over a window of7), Emini surface probability plots, Karplus-Schulz chain flexibilityplots, Jameson-Wolf antigenic index plots, and both Chou-Fasman andGarnier-Osguthorpe-Robson secondary structure plots for the GapC proteinisolated from S. uberis.

FIG. 11 shows Kyte-Doolittle hydropathy plots (averaged over a window of7), Emini surface probability plots, Karplus-Schulz chain flexibilityplots, Jameson-Wolf antigenic index plots, and both Chou-Fasman andGarnier-Osguthorpe-Robson secondary structure plots for the GapC proteinisolated from S. parauberis.

FIG. 12 shows Kyte-Doolittle hydropathy plots (averaged over a window of7), Emini surface probability plots, Karplus-Schulz chain flexibilityplots, Jameson-Wolf antigenic index plots, and both Chou-Fasman andGarnier-Osguthorpe-Robson secondary structure plots for the GapC proteinisolated from S. iniae.

FIG. 13 is a diagrammatic representation of the Chou-Fasman secondarystructure plots for the GapC protein isolated from S. dysgal.

FIG. 14 is a diagrammatic representation of the Chou-Fasman secondarystructure plots for the GapC protein isolated from S. agalactiae.

FIG. 15 is a diagrammatic representation of the Chou-Fasman secondarystructure plots for the GapC protein isolated from S. uberis.

FIG. 16 is a diagrammatic representation of the Chou-Fasman secondarystructure plots for the GapC protein isolated from S. parauberis.

FIG. 17 is a diagrammatic representation of the Chou-Fasman secondarystructure plots for the GapC protein isolated from S. iniae.

FIG. 18 shows the results of SDS-polyacrylamide gel electrophoresis ofrecombinant S. dysgalactiae GapC produced in E. coli DE3. Lane 1,molecular weight markers (20.5-103 kDa; BioRad, Emeryville, Calif.);lane 2, soluble recombinant S. dysgalactiae GapC purified by Ni-NTAaffinity chromatography. Numbers on the left of the figure indicate thepositions of the molecular weight markers (in kDa).

FIG. 19 is a histograph which compares the enzymatic activity of bovineplasmin bound to intact S. dysgalactiae cells; bovine plasmin bound toaffinity-purified recombinant S. dysgalactiae GapC protein; intact S.dysgalactiae cells alone; affinity-purified recombinant S. dysgalactiaeGapC protein alone; and bovine plasmin alone. Activity was determined byan increase in the absorbance at 405 nm following release ofparanitroanalide from the synthetic substrate chromozine-PL (RocheDiagnostics, Laval, Quebec, Canada). The data represents the mean ofthree individual assays.

FIG. 20 compares the change in the percentage of udder quarters infectedwith S. dysgalactiae over a 7 day period among three experimentalgroups: (1) unvaccinated control animals; (2) animals vaccinated withthe Mig Fc binding protein; and (3) animals vaccinated with GapC.Infection was defined as recovery of >500 cfu of the S. dysgalactiae perml of milk secretions.

FIG. 21 plots the maximum number of S. dysgalactiae in any udder quarteragainst serum anti-GapC antibody titre (expressed as the reciprocal ofthe dilution showing activity above background levels). Serum anti-GapCtiters were shown to correlate with the maximum number of cfu/mlrecovered from the mammary glands ®=0.74, as determined using GraphPadPrism software, v. 2.01, from GraphPad Software, Inc., San Diego,Calif.).

FIG. 22 plots cumulative number of infected mammary quarters againstserum antibody titer (again expressed as the reciprocal of the dilutionshowing activity above background levels). A strong correlation betweenthe anti-GapC antibody serum antibody level and the total number ofinfected quarters was observed.

FIG. 23 illustrates the recovery of bacteria from GapC-immunizedanimals; data points are plotted as mean bacterial recoveries/ml milkvs. time (in days post-challenge). In the figure, diamonds (-♦-)represent non-vaccinated animals; squares (-▪-) represent low titeranimals (i.e., animals exhibiting the poorest response against GapC interms of antibody titre), and triangles (-Δ-) represent high titeranimals (i.e., the remaining animals).

FIG. 24 shows recovery of S. dysgalactiae from GapC-immunized animals,plotted as the percent of mammary quarters infected vs. time in dayspost-challenge. In the figure, the stippled bars representnon-vaccinated animals; the cross-hatched bars represent low-titeranimals (i.e., animals exhibiting the poorest response against GapC interms of antibody titre) and the unshaded bars represent high-titeranimals (i.e., the remaining animals).

FIG. 25 depicts the observed inflammatory response to infection with S.dysgalactiae plotted as mean somatic cell counts (SCC) for eachexperimental group versus time in days post challenge. In the figure,diamonds (-♦-) represent unchallenged, unvaccinated quarters, squares(-▪-) represent challenged, unvaccinated animals, triangles (-Δ-)represent challenged, Mig-vaccinated animals, and x's (-X-) representchallenged, GapC-vaccinated animals.

FIG. 26 illustrates somatic cell counts per mammary quarter on day 1post-challenge. In the figure, the bar represents the mean for eachgroup. Squares (-▪-) represent unvaccinated animals; triangles (-▴-)represent GapC-vaccinated animals, and inverted triangles (-▾-)represent Mig-vaccinated animals.

FIG. 27 depicts the somatic cell counts of non-vaccinated,non-challenged and GapC-immunized, high-titer animals (i.e., the fouranimals exhibiting the highest antibody titres of the eight animals inthe particular group) for seven days post challenge, plotted as thelog₁₀ of the mean somatic cell count/ml milk against time in days postchallenge. Diamonds (-♦-) represent unchallenged, unvaccinated animals,squares (-▪-) represent challenged, unvaccinated animals, and triangles(-Δ-) represent challenged, GapC-vaccinated animals.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology, and immunology, which are within the skillof the art. Such techniques are explained fully in the literature. See,e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A LaboratoryManual, Vols. I, II and III, Second Edition (1989); Perbal, B., APractical Guide to Molecular Cloning (1984); the series, Methods InEnzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); andHandbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.Blackwell eds., 1986, Blackwell Scientific Publications).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

The following amino acid abbreviations are used throughout the text:

Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid:Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu (E)Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L)Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro(P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr(Y) Valine: Val (V)

1. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a Streptococcus GapC protein” includes a mixture of two ormore such proteins, and the like.

The terms “GapC protein” and “GapC plasmin binding protein” (usedinterchangeably herein) or a nucleotide sequence encoding the same,intends a protein or a nucleotide sequence, respectively, which isderived from a GapC gene found in a variety of Streptococcus species,including, without limitation certain strains of group A streptococci(Lottenbery, R., et al., (1987) Infect. Immun. 55:1914-1918). Thenucleotide sequence of representative Streptococcus gapC genes, and thecorresponding amino acid sequence of the GapC proteins encoded by thesegenes, are depicted in the Figures. In particular, FIGS. 1 through 5depict the isolated nucleotide sequences and isolated amino acidsequences of S. dysgalactiae (SEQ ID NO:3 and SEQ ID NO:4,respectively), S. agalactiae (SEQ ID NO:5 and SEQ ID NO:6,respectively), S. uberis (SEQ ID NO:7 and SEQ ID NO:8, respectively), S.parauberis (SEQ ID NO:9 and SEQ ID NO:10, respectively), and S. iniae(SEQ ID NO:11 and SEQ ID NO:12, respectively). However, a GapC proteinas defined herein is not limited to the depicted sequences as subtypesof each of these Streptococcus species are known and variations in GapCproteins will occur between them.

Representative gapC genes, derived from S. dysgalactiae, S. agalactiae,S. uberis, and S. parauberis, are found in the plasmids pET15bgapC,pMF521c pMF521a, pMF521d, and pMF521e, respectively.

Furthermore, the derived protein or nucleotide sequences need not bephysically derived from the gene described above, but may be generatedin any manner, including for example, chemical synthesis, isolation(e.g., from S. dysgalactiae) or by recombinant production, based on theinformation provided herein. Additionally, the term intends proteinshaving amino acid sequences substantially homologous (as defined below)to contiguous amino acid sequences encoded by the genes, which displayimmunological and/or plasmin-binding activity.

Thus, the terms intend full-length, as well as immunogenic, truncatedand partial sequences, and active analogs and precursor forms of theproteins. Also included in the term are nucleotide fragments of the genethat include at least about 8 contiguous base pairs, more preferably atleast about 10-20 contiguous base pairs, and most preferably at leastabout 25 to 50, or more, contiguous base pairs of the gene, or anyintegers between these values. Such fragments are useful as probes andin diagnostic methods, discussed more fully below.

The terms also include those forms possessing, as well as lacking, asignal sequence, if such is present, as well as the nucleic acidsequences coding therefore. Additionally, the term intends forms of theGapC proteins which lack a membrane anchor region, and nucleic acidsequences encoding proteins with such deletions. Such deletions may bedesirable in systems that do not provide for secretion of the protein.Furthermore, the plasmin-binding domains of the proteins, may or may notbe present. Thus, for example, if the GapC plasmin-binding protein willbe used to purify plasmin, the plasmin-binding domain will generally beretained. If the protein is to be used in vaccine compositions,immunogenic epitopes which may or may not include the plasmin-bindingdomain, will be present.

The terms also include proteins in neutral form or in the form of basicor acid addition salts depending on the mode of preparation. Such acidaddition salts may involve free amino groups and basic salts may beformed with free carboxyls. Pharmaceutically acceptable basic and acidaddition salts are discussed further below. In addition, the proteinsmay be modified by combination with other biological materials such aslipids (both those occurring naturally with the molecule or other lipidsthat do not destroy immunological activity) and saccharides, or by sidechain modification, such as acetylation of amino groups, phosphorylationof hydroxyl side chains, oxidation of sulfhydryl groups, glycosylationof amino acid residues, as well as other modifications of the encodedprimary sequence.

The term therefore intends deletions, additions and substitutions to thesequence, so long as the polypeptide functions to produce animmunological response as defined herein. In this regard, particularlypreferred substitutions will generally be conservative in nature, i.e.,those substitutions that take place within a family of amino acids. Forexample, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cystine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, or viceversa; an aspartate with a glutamate or vice versa; a threonine with aserine or vice versa; or a similar conservative replacement of an aminoacid with a structurally related amino acid, will not have a majoreffect on the biological activity. Proteins having substantially thesame amino acid sequence as the reference molecule, but possessing minoramino acid substitutions that do not substantially affect theimmunogenicity and/or plasmin-binding affinity of the protein, aretherefore within the definition of the reference polypeptide.

For example, the polypeptide of interest may include up to about 5-10conservative or non-conservative amino acid substitutions, or even up toabout 15-25 or 20-50 conservative or non-conservative amino acidsubstitutions, or any integer between these values, so long as thedesired function of the molecule remains intact.

In this regard, GapC proteins isolated from streptococci exhibit severalvariable regions in their amino acid sequences, located at amino acidpositions 62 to 81; 102 to 112; 165 to 172; 248 to 271; and 286 to 305.These regions, which in S. dysgalactiae, S. agalactiae, S. uberis, S.parauberis and S. iniae exhibit from 1 to 9 amino acid substitutions,are likely to be amenable to variation without substantially affectingimmunogenic or enzymatic function.

Similarly, substitutions occurring in the transmembrane binding domain,if present, and the signal sequence, if present, normally will notaffect immunogenicity. One of skill in the art may readily determineother regions of the molecule of interest that can tolerate change byreference to the protein structure data shown in FIGS. 8-17 herein.

The term “streptococcal GapC protein” intends a GapC plasmin-bindingprotein, as defined above, derived from a streptococcal species thatproduces the same, including, but not limited to S. dysgalactiae, S.agalactiae, S. uberis, S. parauberis, and S. iniae. For example, a “S.dysgalactiae GapC protein” is a GapC plasmin-binding protein as definedabove, derived from S. dysgalactiae. Similarly, an “S. agalactiae GapCprotein” intends a gapc binding protein derived from S. agalactiae.

“Wild type” or “native” proteins or polypeptides refer to proteins orpolypeptides isolated from the source in which the proteins naturallyoccur. “Recombinant” polypeptides refer to polypeptides produced byrecombinant DNA techniques; i.e., produced from cells transformed by anexogenous DNA construct encoding the desired polypeptide.

“Synthetic” polypeptides are those prepared by chemical synthesis.

An “isolated” protein or polypeptide is a protein or polypeptidemolecule separate and discrete from the whole organism with which themolecule is found in nature; or a protein or polypeptide devoid, inwhole or part, of sequences normally associated with it in nature; or asequence, as it exists in nature, but having heterologous sequences (asdefined below) in association therewith.

The term “functionally equivalent” intends that the amino acid sequenceof a GapC plasmin-binding protein is one that will elicit asubstantially equivalent or enhanced immunological response, as definedabove, as compared to the response elicited by a GapC plasmin-bindingprotein having identity with the reference GapC plasmin-binding protein,or an immunogenic portion thereof.

The term “epitope” refers to the site on an antigen or hapten to whichspecific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite.” Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen.

The terms “immunogenic” protein or polypeptide refer to an amino acidsequence which elicits an immunological response as described above. An“immunogenic” protein or polypeptide, as used herein, includes thefull-length sequence of the GapC plasmin-binding protein in question,with or without the signal sequence, membrane anchor domain and/orplasmin-binding domain, analogs thereof, or immunogenic fragmentsthereof. By “immunogenic fragment” is meant a fragment of a GapCplasmin-binding protein which includes one or more epitopes and thuselicits the immunological response described above. Such fragments canbe identified using any number of epitope mapping techniques, well knownin the art. See, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J.For example, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Similarly,conformational epitopes are readily identified by determining spatialconformation of amino acids such as by, e.g., x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra. Antigenic regions of proteins can also be identifiedusing standard antigenicity and hydropathy plots, such as thosecalculated using, e.g., the Omiga version 1.0 software program availablefrom the Oxford Molecular Group. This computer program employs theHopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981)78:3824-3828 for determining antigenicity profiles, and theKyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132for hydropathy plots. FIGS. 8 to 12 herein depict Kyte-Doolittleprofiles for representative proteins encompassed by the invention.

Immunogenic fragments, for purposes of the present invention, willusually include at least about 3 amino acids, preferably at least about5 amino acids, more preferably at least about 10-15 amino acids, andmost preferably 25 or more amino acids, of the parent GapCplasmin-binding-binding protein molecule. There is no critical upperlimit to the length of the fragment, which may comprise nearly thefull-length of the protein sequence, or even a fusion protein comprisingtwo or more epitopes of GapC.

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest.

By “subunit vaccine composition” is meant a composition containing atleast one immunogenic polypeptide, but not all antigens, derived from orhomologous to an antigen from a pathogen of interest. Such a compositionis substantially free of intact pathogen cells or particles, or thelysate of such cells or particles. Thus, a “subunit vaccine composition”is prepared from at least partially purified (preferably substantiallypurified) immunogenic polypeptides from the pathogen, or recombinantanalogs thereof. A subunit vaccine composition can comprise the subunitantigen or antigens of interest substantially free of other antigens orpolypeptides from the pathogen.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual in a formulationor composition without causing any undesirable biological effects orinteracting in a deleterious manner with any of the components of thecomposition in which it is contained.

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to the composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells and/or γδ T cells,directed specifically to an antigen or antigens included in thecomposition or vaccine of interest. Preferably, the host will displayeither a therapeutic or protective immunological response such thatresistance of the mammary gland to new infection will be enhanced and/orthe clinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms normallydisplayed by an infected host and/or a quicker recovery time.

By “nucleic acid immunization” is meant the introduction of a nucleicacid molecule encoding one or more selected antigens into a host cell,for the in vivo expression of an antigen, antigens, an epitope, orepitopes. The nucleic acid molecule can be introduced directly into arecipient subject, such as by injection, inhalation, oral, intranasaland mucosal administration, or the like, or can be introduced ex vivo,into cells which have been removed from the host. In the latter case,the transformed cells are reintroduced into the subject where an immuneresponse can be mounted against the antigen encoded by the nucleic acidmolecule.

The term “treatment” as used herein refers to either (1) the preventionof infection or reinfection (prophylaxis), or (2) the reduction orelimination of symptoms of the disease of interest (therapy).

By “mastitis” is meant an inflammation of the mammary gland in mammals,including in cows, ewes, goats, sows, mares, and the like, caused by thepresence of S. uberis. The infection manifests itself by theinfiltration of phagocytic cells in the gland. Generally, 4 clinicaltypes of mastitis are recognized: (1) peracute, associated withswelling, heat, pain, and abnormal secretion in the gland andaccompanied by fever and other signs of systemic disturbance, such asmarked depression, rapid weak pulse, sunken eyes, weakness and completeanorexia; (2) acute, with changes in the gland similar to those abovebut where fever, anorexia and depression are slight to moderate; (3)subacute, where no systemic changes are displayed and the changes in thegland and its secretion are less marked: and (4) subclinical, where theinflammatory reaction is detectable only by standard tests for mastitis.

Standard tests for the detection of mastitis include but are not limitedto, the California Mastitis Test, the Wisconsin Mastitis Test, theNagase test, the electronic cell count and somatic cell counts used todetect a persistently high white blood cell content in milk. In general,a somatic cell count of about 300,000 to about 500,000 cells per ml orhigher, in milk will indicate the presence of infection. Thus, a vaccineis considered effective in the treatment and/or prevention of mastitiswhen, for example, the somatic cell count in milk is retained belowabout 500,000 cells per ml. For a discussion of mastitis and thediagnosis thereof, see, e.g., The Merck Veterinary Manual: A Handbook ofDiagnosis, Therapy, and Disease Prevention and Control for theVeterinarian, Merck and Co., Rahway, N.J., 1991.

By the terms “vertebrate,” “subject,” and “vertebrate subject” are meantany member of the subphylum Chordata, including, without limitation,mammals such as cattle, sheep, pigs, goats, horses, and humans; domesticanimals such as dogs and cats; and birds, including domestic, wild andgame birds such as cocks and hens including chickens, turkeys and othergallinaceous birds; and fish. The term does not denote a particular age.Thus, both adult and newborn animals, as well as fetuses, are intendedto be covered.

A “nucleic acid” molecule can include, but is not limited to,procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA,genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA.

An “isolated” nucleic acid molecule is a nucleic acid molecule separateand discrete from the whole organism with which the molecule is found innature; or a nucleic acid molecule devoid, in whole or part, ofsequences normally associated with it in nature; or a sequence, as itexists in nature, but having heterologous sequences (as defined below)in association therewith. The term “isolated” in the context of apolynucleotide intends that the polynucleotide is isolated from thechromosome with which it is normally associated, and is isolated fromthe complete genomic sequence in which it normally occurs.

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell-known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

A “coding sequence” or a “nucleotide sequence encoding” a particularprotein, is a nucleotide sequence which is transcribed and translatedinto a polypeptide in vitro or in vivo when placed under the control ofappropriate regulatory elements. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, procaryotic sequences, cDNA fromeucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian)DNA, and even synthetic DNA sequences. A transcription terminationsequence will usually be located 3′ to the coding sequence. A“complementary” sequence is one in which the nitrogenous base at a givennucleotide position is the complement of the nitrogenous base appearingat the same position in the reference sequence. To illustrate, thecomplement of adenosine is tyrosine, and vice versa; similarly, cytosineis complementary to guanine, and vice versa; hence, the complement ofthe reference sequence 5′-ATGCTGA-3′ would be 5′-TACGACT-3′.

A “wild-type” or “native” sequence, as used herein, refers topolypeptide encoding sequences that are essentially as they are found innature, e.g., the S. dysgalactiae GapC protein encoding sequencesdepicted in FIGS. 1A-1B (SEQ ID NO:4).

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature; and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “recombinant” as used withrespect to a protein or polypeptide means a polypeptide produced byexpression of a recombinant polynucleotide. “Recombinant host cells,”“host cells,” “cells,” “cell lines,” “cell cultures,” and other suchterms denoting procaryotic microorganisms or eucaryotic cell linescultured as unicellular entities, are used interchangeably, and refer tocells which can be, or have been, used as recipients for recombinantvectors or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellwhich are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a desired peptide, are included in the progeny intended by thisdefinition, and are covered by the above terms.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when the sequences exhibit atleast about 80%-85%, preferably at least about 90%, and most preferablyat least about 95%-98% sequence identity over a defined length of themolecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5Suppl. 3:353-358, National biomedical Research Foundation, Washington,D.C., which adapts the local homology algorithm of Smith and Waterman(1981) Advances in Appl. Math. 2:482-489 for peptide analysis. Programsfor determining nucleotide sequence identity are available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAPprograms, which also rely on the Smith and Waterman algorithm. Theseprograms are readily utilized with the default parameters recommended bythe manufacturer and described in the Wisconsin Sequence AnalysisPackage referred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUTM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: www.ncbi.nlm.gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

By the term “degenerate variant” is intended a polynucleotide containingchanges in the nucleic acid sequence thereof, that encodes a polypeptidehaving the same amino acid sequence as the polypeptide encoded by thepolynucleotide from which the degenerate variant is derived.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

A “heterologous” region of a DNA construct is an identifiable segment ofDNA within or attached to another DNA molecule that is not found inassociation with the other molecule in nature. Thus, when theheterologous region encodes a bacterial gene, the gene will usually beflanked by DNA that does not flank the bacterial gene in the genome ofthe source bacteria. Another example of the heterologous coding sequenceis a construct where the coding sequence itself is not found in nature(e.g., synthetic sequences having codons different from the nativegene). Allelic variation or naturally occurring mutational events do notgive rise to a heterologous region of DNA, as used herein.

A “vector” is a replicon, such as a plasmid, phage, or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A vector is capable of transferring genesequences to target cells (e.g., bacterial plasmid vectors, viralvectors, non-viral vectors, particulate carriers, and liposomes).Typically, the terms “vector construct,” “expression vector,” “geneexpression vector,” “gene delivery vector,” “gene transfer vector,” and“expression cassette” all refer to an assembly which is capable ofdirecting the expression of a sequence or gene of interest. Thus, theterms include cloning and expression vehicles, as well as viral vectors.

These assemblies include a promoter which is operably linked to thesequences or gene(s) of interest. Other control elements may be presentas well. The expression cassettes described herein may be containedwithin a plasmid construct. In addition to the components of theexpression cassette, the plasmid construct may also include a bacterialorigin of replication, one or more selectable markers, a signal whichallows the plasmid construct to exist as single-stranded DNA (e.g., aM13 origin of replication), a multiple cloning site, and a “mammalian”origin of replication (e.g., a SV40 or adenovirus origin ofreplication).

DNA “control elements” refers collectively to transcription promoters,transcription enhancer elements, transcription termination sequences,polyadenylation sequences (located 3′ to the translation stop codon),sequences for optimization of initiation of translation (located 5′ tothe coding sequence), translation termination sequences, upstreamregulatory domains, ribosome binding sites and the like, whichcollectively provide for the transcription and translation of a codingsequence in a host cell. See e.g., McCaughan et al. (1995) PNAS USA92:5431-5435; Kochetov et al (1998) FEBS Letts. 440:351-355. Not all ofthese control sequences need always be present in a recombinant vectorso long as the desired gene is capable of being transcribed andtranslated.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter and the coding sequence and the promoter canstill be considered “operably linked” to the coding sequence. Similarly,“control elements compatible with expression in a subject” are thosewhich are capable of effecting the expression of the coding sequence inthat subject.

A control element, such as a promoter, “directs the transcription” of acoding sequence in a cell when RNA polymerase will bind the promoter andtranscribe the coding sequence into mRNA, which is then translated intothe polypeptide encoded by the coding sequence.

A “host cell” is a cell which has been transformed, or is capable oftransformation, by an exogenous nucleic acid molecule.

A cell has been “transformed” by exogenous DNA when such exogenous DNAhas been introduced inside the cell membrane. Exogenous DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In procaryotes and yeasts, for example, theexogenous DNA may be maintained on an episomal element, such as aplasmid. With respect to eucaryotic cells, a stably transformed cell isone in which the exogenous DNA has become integrated into the chromosomeso that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeucaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a subject, including but not limited to, forexample, blood, plasma, serum, fecal matter, urine, bone marrow, bile,spinal fluid, lymph fluid, samples of the skin, external secretions ofthe skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, blood cells, organs, biopsies and also samples of in vitrocell culture constituents including but not limited to conditioned mediaresulting from the growth of cells and tissues in culture medium, e.g.,recombinant cells, and cell components.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes,metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.The term “fluorescer” refers to a substance or a portion thereof whichis capable of exhibiting fluorescence in the detectable range.Particular examples of labels which may be used under the inventioninclude fluorescein, rhodamine, dansyl, umbelliferone, Texas red,luminol, NADPH and α-β-galactosidase.

2. Modes of Carrying out the Invention

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

Central to the present invention is the discovery that the GapC proteinis capable of eliciting an immune response in a vertebrate subject. Inparticular, the genes for the GapC protein in S. dysgalactiae, S.agalactiae, S. uberis, S. parauberis, and S. iniae have been isolated,sequenced and characterized, and the protein sequences for those geneshave been deduced therefrom. The complete DNA sequences for those genesand the corresponding amino acid sequences are shown in FIGS. 1 through5.

As described in the examples, the full-length S. dysgalactiae gapC gene,depicted at nucleotide positions 1-1011, inclusive, of FIGS. 1A-1B,encodes the full-length S. dysgalactiae GapC protein of 336 amino acids,shown as amino acids 1-336, inclusive, of the same figure. S.dysgalactiae GapC has a predicted molecular weight of about 36 kDa.(calculated using the Peptide Sort program of the GCG Wisconsin Package,version 10, provided by the SeqWeb sequence analysis package, version1.1 of the Canadian Bioinformatics Resource). Similarly, the gapC genesisolated from S. agalactiae, S. uberis, S. parauberis and S. iniae aredepicted in FIGS. 2 through 5; each encodes a full-length GapC proteinalso of 336 amino acids, each also having a predicted molecular weightof about 36 kDa. None of the full-length sequences appear to include asignal peptide or a membrane anchor region.

FIGS. 6 and 7 show an alignment of DNA and amino acid sequences,respectively, showing regions of homology and variability that existamong GapC proteins from various streptococci strains. In particular,several variable regions are located at amino acid positions 62 to 81;102 to 112; 165 to 172; 248 to 271; and 286 to 305. Such variableregions are typically more amenable to change. Hence, amino acid changesin these regions, such as substitutions, additions and deletions, arelikely tolerated.

FIGS. 8 through 12 show plots of the following for each of the GapCproteins of the present invention: Kyte-Doolittle hydrophathy, averagedover a window of 7; surface probability according to Emini; chainflexibility according to Karplus-Schulz; antigenicity index according toJameson-Wolf; secondary structure according to Gamier-Osguthorpe-Robson;secondary structure according to Chou-Fasman; and predictedglycosylation sites. FIGS. 13 through 17 show plots of secondarystructure according to Chou-Fasman for each of the GapC proteins of thepresent invention. One of skill in the art can readily use theinformation presented in FIGS. 8 to 17 to determine immunogenic regionsin the protein for use in vaccine compositions.

S. dysgalactiae GapC plasmin-binding protein, immunogenic fragmentsthereof or chimeric proteins including the same, can be provided insubunit vaccine compositions. In addition to use in vaccinecompositions, the proteins or antibodies thereto can be used asdiagnostic reagents to detect the presence of infection in a vertebratesubject. Similarly, the genes encoding the proteins can be cloned andused to design probes to detect and isolate homologous genes in otherbacterial strains. For example, fragments comprising at least about15-20 nucleotides, more preferably at least about 20-50 nucleotides, andmost preferably about 60-100 nucleotides, or any integer between thesevalues, will find use in these embodiments.

The vaccine compositions of the present invention can be used to treator prevent a wide variety of bacterial infections in vertebratesubjects. For example, vaccine compositions including GapCplasmin-binding proteins from S. dysgalactiae, S. uberis, S. parauberis,S. iniae, and/or group B streptococci (GBS) such as S. agalactiae, canbe used to treat streptococcal infections in vertebrate subjects thatare caused by these or other species. In particular, S. uberis and S.agalactiae are common bacterial pathogens associated with mastitis inbovine, equine, ovine and goat species. Additionally, group Bstreptococci, such as S. agalactiae, are known to cause numerous otherinfections in vertebrates, including septicemia, meningitis, bacteremia,impetigo, arthritis, urinary tract infections, abscesses, spontaneousabortion etc. Hence, vaccine compositions containing streptococcal GapCplasmin-binding proteins will find use in treating and/or preventing awide variety of streptococcal infections.

Similarly, GapC plasmin-binding proteins derived from other bacterialgenera such as Staphylococcus, Mycobacterium, Escherichia, Pseudomonas,Nocardia, Pasteurella, Clostridium and Mycoplasma will find use fortreating bacterial infections caused by species belonging to thosegenera. Thus, it is readily apparent that GapC plasmin-binding proteinscan be used to treat and/or prevent a wide variety of bacterialinfections in numerous species.

The streptococcal GapC plasmin-binding proteins of the present inventioncan be used in vaccine compositions either alone or in combination withother bacterial, fungal, viral or protozoal antigens. These antigens canbe provided separately or even as fusion proteins comprising one or moreepitopes of a GapC plasmin-binding protein fused to one or more of theseantigens. For example, other immunogenic proteins from S. uberis, suchas the CAMP factor, hyaluronic acid capsule, hyaluronidase, R-likeprotein and plasminogen activator, can be administered with the GapCprotein. Additionally, immunogenic proteins from other organismsinvolved in mastitis, such as from the genera Staphylococcus,Corynebacterium, Pseudomonas, Nocardia, Clostridium, Mycobacterium,Mycoplasma, Pasteurella, Prototheca, other streptococci, coliformbacteria, as well as yeast, can be administered along with the GapCplasmin-binding proteins described herein to provide a broad spectrum ofprotection. Thus, for example, immunogenic proteins from Staphylococcusaureus, Str. agalactiae, Str. dysgalactiae, Str. zooepidemicus,Corynebacterium pyogenes, Pseudomonas aeruginosa, Nocardia asteroides,Clostridium perfringens, Escherichia coli, Enterobacter aerogenes andKlebsiella spp. can be provided along with the GapC plasmin-bindingproteins of the present invention.

Additionally, GapC proteins from different streptococcal species may beused together in the vaccine compositions of the present invention. Inthis embodiment, the multiple GapC proteins may be provided as fusionproteins or as discrete antigens in the same or different vaccinecompositions.

Production of GapC Plasmin-Binding Proteins

The above-described plasmin-binding proteins and active fragments,analogs and chimeric proteins derived from the same, can be produced byvariety of methods. Specifically, GapC plasmin-binding proteins can beisolated directly from bacteria which express the same. This isgenerally accomplished by first preparing a crude extract which lackscellular components and several extraneous proteins. The desiredproteins can then be further purified from the cell lysate fraction by,e.g., column chromatography, HPLC, immunoadsorbent techniques or otherconventional methods well known in the art.

More particularly, techniques for isolating GapC plasmin-bindingproteins have been described. For example, the GapC protein of S.pyogenes was purified from a crude cell extract by precipitation withammonium sulfate, followed by two cycles of chromatography through aMono FPLC column, and single cycles through superose 12 FPLC, andTSK-phenol HPLC columns (Pancholi, V. and Fischetti, V A (1992) J Exptl.Med 76:415-426). Another technique involves the use of a NAD⁺-agaroseaffinity column to purify GapC from lysed protoplasts of S. pyogenesstrain 64/14 (Winram, S B and Lottenberg, R (1996) Microbiol.142:2311-2320).

Alternatively, the proteins can be recombinantly produced as describedherein. As explained above, these recombinant products can take the formof partial protein sequences, full-length sequences, precursor formsthat include signal sequences, mature forms without signals, or evenfusion proteins (e.g., with an appropriate leader for the recombinanthost, or with another subunit antigen sequence for Streptococcus oranother pathogen).

In one embodiment of the present invention, the GapC proteins are fusedto a histidine tag, produced by recombinant means, and are then purifiedfrom a cell lysate fraction using affinity chromatography. See Example1A-E, infra.

The GapC plasmin-binding proteins of the present invention can beisolated based on the ability of the protein products to bind plasmin,using plasmin-binding assays as described below. See, e.g., the methoddescribed in section F.3. of Example 1, infra. Thus, gene libraries canbe constructed and the resulting clones used to transform an appropriatehost cell. Colonies can be pooled and screened for clones havingplasmin-binding activity. Colonies can also be screened using polyclonalserum or monoclonal antibodies to the plasmin-binding protein.

Alternatively, once the amino acid sequences are determined,oligonucleotide probes which contain the codons for a portion of thedetermined amino acid sequences can be prepared and used to screengenomic or cDNA libraries for genes encoding the subject proteins. Thebasic strategies for preparing oligonucleotide probes and DNA libraries,as well as their screening by nucleic acid hybridization, are well knownto those of ordinary skill in the art. See, e.g., DNA Cloning: Vol. 1I,supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis,supra; Sambrook et al., supra. Once a clone from the screened libraryhas been identified by positive hybridization, it can be confirmed byrestriction enzyme analysis and DNA sequencing that the particularlibrary insert contains GapC plasmin-binding protein gene or a homologthereof. The genes can then be further isolated using standardtechniques and, if desired, PCR approaches or restriction enzymesemployed to delete portions of the full-length sequence.

Similarly, genes can be isolated directly from bacteria using knowntechniques, such as phenol extraction and the sequence furthermanipulated to produce any desired alterations. See, e.g., Sambrook etal., supra, for a description of techniques used to obtain and isolateDNA.

Alternatively, DNA sequences encoding the proteins of interest can beprepared synthetically rather than cloned. The DNA sequences can bedesigned with the appropriate codons for the particular amino acidsequence. In general, one will select preferred codons for the intendedhost if the sequence will be used for expression. The complete sequenceis assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge(1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay etal. (1984) J. Biol. Chem. 259:6311.

Once coding sequences for the desired proteins have been prepared orisolated, they can be cloned into any suitable vector or replicon.Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, Sambrook et al., supra; DNA Cloning, supra; B.Perbal, supra.

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired protein is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. If a signal sequence is included, it caneither be the native, homologous sequence, or a heterologous sequence.For example, the signal sequence for S. dysgalactiae GapCplasmin-binding protein can be used for secretion thereof, as can anumber of other signal sequences, well known in the art. Leadersequences can be removed by the host in post-translational processing.See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.

Other regulatory sequences may also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Regulatory sequences are known to those of skill inthe art, and examples include those which cause the expression of a geneto be turned on or off in response to a chemical or physical stimulus,including the presence of a regulatory compound. Other types ofregulatory elements may also be present in the vector, for example,enhancer sequences.

The control sequences and other regulatory sequences may be ligated tothe coding sequence prior to insertion into a vector, such as thecloning vectors described above. Alternatively, the coding sequence canbe cloned directly into an expression vector which already contains thecontrol sequences and an appropriate restriction site.

In some cases it may be necessary to modify the coding sequence so thatit may be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. It may also bedesirable to produce mutants or analogs of the GapC plasmin-bindingprotein. Mutants or analogs may be prepared by the deletion of a portionof the sequence encoding the protein, by insertion of a sequence, and/orby substitution of one or more nucleotides within the sequence.Techniques for modifying nucleotide sequences, such as site-directedmutagenesis, are described in, e.g., Sambrook et al., supra; DNACloning, supra; Nucleic Acid Hybridization, supra.

The expression vector is then used to transform an appropriate hostcell. A number of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Madin-Darby bovine kidney (“MDBK”) cells, as well as others. Similarly,bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcusspp., will find use with the present expression constructs. Yeast hostsuseful in the present invention include inter alia, Saccharomycescerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha,Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii,Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.Insect cells for use with baculovirus expression vectors include, interalia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni.

Depending on the expression system and host selected, the proteins ofthe present invention are produced by culturing host cells transformedby an expression vector described above under conditions whereby theprotein of interest is expressed. The protein is then isolated from thehost cells and purified. If the expression system secretes the proteininto the growth media, the protein can be purified directly from themedia. If the protein is not secreted, it is isolated from cell lysates.The selection of the appropriate growth conditions and recovery methodsare within the skill of the art.

The proteins of the present invention may also be produced by chemicalsynthesis such as solid phase peptide synthesis, using known amino acidsequences or amino acid sequences derived from the DNA sequence of thegenes of interest. Such methods are known to those skilled in the art.See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis,2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984) and G. Barany and R.B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E.Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp.3-254, for solid phase peptide synthesis techniques; and M. Bodansky,Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E.Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis,Biology, supra, Vol. 1, for classical solution synthesis. Chemicalsynthesis of peptides may be preferable if a small fragment of theantigen in question is capable of raising an immunological response inthe subject of interest.

The GapC plasmin-binding proteins of the present invention, or theirfragments, can be used to produce antibodies, both polyclonal andmonoclonal. If polyclonal antibodies are desired, a selected mammal,(e.g., mouse, rabbit, goat, horse, etc.) is immunized with an antigen ofthe present invention, or its fragment, or a mutated antigen. Serum fromthe immunized animal is collected and treated according to knownprocedures. See, e.g., Jurgens et al. (1985) J. Chrom. 348:363-370. Ifserum containing polyclonal antibodies is used, the polyclonalantibodies can be purified by immunoaffinity chromatography, using knownprocedures.

Monoclonal antibodies to the GapC plasmin-binding proteins and to thefragments thereof, can also be readily produced by one skilled in theart. The general methodology for making monoclonal antibodies by usinghybridoma technology is well known. Immortal antibody-producing celllines can be created by cell fusion, and also by other techniques suchas direct transformation of B lymphocytes with oncogenic DNA, ortransfection with Epstein-Barr virus. See, e.g., M. Schreier et al.,Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodiesand T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies(1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783;4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890.Panels of monoclonal antibodies produced against the GapCplasmin-binding proteins, or fragments thereof, can be screened forvarious properties; i.e., for isotype, epitope, affinity, etc.Monoclonal antibodies are useful in purification, using immunoaffinitytechniques, of the individual antigens which they are directed against.Both polyclonal and monoclonal antibodies can also be used for passiveimmunization or can be combined with subunit vaccine preparations toenhance the immune response. Polyclonal and monoclonal antibodies arealso useful for diagnostic purposes.

Vaccine Formulations and Administration

The GapC plasmin-binding proteins of the present invention can beformulated into vaccine compositions, either alone or in combinationwith other antigens, for use in immunizing subjects as described below.Methods of preparing such formulations are described in, e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 18 Edition, 1990. Typically, the vaccines of the present inventionare prepared as injectables, either as liquid solutions or suspensions.Solid forms suitable for solution in or suspension in liquid vehiclesprior to injection may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in liposome vehicles.The active immunogenic ingredient is generally mixed with a compatiblepharmaceutical vehicle, such as, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents and pH bufferingagents.

Adjuvants which enhance the effectiveness of the vaccine may also beadded to the formulation. Adjuvants may include for example, muramyldipeptides, avridine, aluminum hydroxide, dimethyldioctadecyl ammoniumbromide (DDA), oils, oil-in-water emulsions, saponins, cytokines, andother substances known in the art.

The GapC plasmin-binding protein may be linked to a carrier in order toincrease the immunogenicity thereof. Suitable carriers include large,slowly metabolized macro-molecules such as proteins, including serumalbumins, keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art; polysaccharides, such as sepharose, agarose, cellulose,cellulose beads and the like; polymeric amino acids such as polyglutamicacid, polylysine, and the like; amino acid copolymers; and inactivevirus particles.

The GapC plasmin-binding proteins may be used in their native form ortheir functional group content may be modified by, for example,succinylation of lysine residues or reaction with Cys-thiolactone. Asulfhydryl group may also be incorporated into the carrier (or antigen)by, for example, reaction of amino functions with 2-iminothiolane or theN-hydroxysuccinimide ester of 3-(4-dithiopyridyl propionate. Suitablecarriers may also be modified to incorporate spacer arms (such ashexamethylene diamine or other bifunctional molecules of similar size)for attachment of peptides.

Other suitable carriers for the GapC plasmin-binding proteins of thepresent invention include VP6 polypeptides of rotaviruses, or functionalfragments thereof, as disclosed in U.S. Pat. No. 5,071,651, incorporatedherein by reference. Also useful is a fusion product of a viral proteinand the subject immunogens made by methods disclosed in U.S. Pat. No.4,722,840. Still other suitable carriers include cells, such aslymphocytes, since presentation in this form mimics the natural mode ofpresentation in the subject, which gives rise to the immunized state.Alternatively, the proteins of the present invention may be coupled toerythrocytes, preferably the subject's own erythrocytes. Methods ofcoupling peptides to proteins or cells are known to those of skill inthe art.

Furthermore, the GapC plasmin-binding proteins (or complexes thereof)may be formulated into vaccine compositions in either neutral or saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the active polypeptides) and whichare formed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from free carboxyl groups may alsobe derived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine, and the like.

Vaccine formulations will contain a “therapeutically effective amount”of the active ingredient, that is, an amount capable of eliciting animmune response in a subject to which the composition is administered.In the treatment and prevention of mastitis, for example, a“therapeutically effective amount” would preferably be an amount thatenhances resistance of the mammary gland to new infection and/or reducesthe clinical severity of the disease. Such protection will bedemonstrated by either a reduction or lack of symptoms normallydisplayed by an infected host, a quicker recovery time and/or a loweredsomatic cell count in milk from the infected quarter. For example, theability of the composition to retain or bring the somatic cell count(SCC) in milk below about 500,000 cells per ml, the threshold value setby the International Dairy Federation, above which, animals areconsidered to have clinical mastitis, will be indicative of atherapeutic effect.

The exact amount is readily determined by one skilled in the art usingstandard tests. The GapC plasmin-binding protein concentration willtypically range from about 1% to about 95% (w/w) of the composition, oreven higher or lower if appropriate. With the present vaccineformulations, 5 to 500 μg of active ingredient per ml of injectedsolution should be adequate to raise an immunological response when adose of 1 to 3 ml per animal is administered.

To immunmze a subject, the vaccine is generally administeredparenterally, usually by intramuscular injection. Other modes ofadministration, however, such as subcutaneous, intraperitoneal andintravenous injection, are also acceptable. The quantity to beadministered depends on the animal to be treated, the capacity of theanimal's immune system to synthesize antibodies, and the degree ofprotection desired. Effective dosages can be readily established by oneof ordinary skill in the art through routine trials establishing doseresponse curves. The subject is immunized by administration of thevaccine in at least one dose, and preferably two doses. Moreover, theanimal may be administered as many doses as is required to maintain astate of immunity to infection.

Additional vaccine formulations which are suitable for other modes ofadministration include suppositories and, in some cases, aerosol,intranasal, oral formulations, and sustained release formulations. Forsuppositories, the vehicle composition will include traditional bindersand carriers, such as, polyalkaline glycols, or triglycerides. Suchsuppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), preferablyabout 1% to about 2%. Oral vehicles include such normally employedexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium, stearate, sodium saccharin cellulose, magnesiumcarbonate, and the like. These oral vaccine compositions may be taken inthe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations, or powders, and contain from about 10% to about95% of the active ingredient, preferably about 25% to about 70%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

Controlled or sustained release formulations are made by incorporatingthe protein into carriers or vehicles such as liposomes, nonresorbableimpermeable polymers such as ethylenevinyl acetate copolymers andHytrel® copolymers, swellable polymers such as hydrogels, or resorbablepolymers such as collagen and certain polyacids or polyesters such asthose used to make resorbable sutures. The GapC plasmin-binding proteinscan also be delivered using implanted mini-pumps, well known in the art.

The GapC plasmin-binding proteins of the instant invention can also beadministered via a carrier virus which expresses the same. Carrierviruses which will find use with the instant invention include but arenot limited to the vaccinia and other pox viruses, adenovirus, andherpes virus. By way of example, vaccinia virus recombinants expressingthe novel proteins can be constructed as follows. The DNA encoding theparticular protein is first inserted into an appropriate vector so thatit is adjacent to a vaccinia promoter and flanking vaccinia DNAsequences, such as the sequence encoding thymidine kinase (TK). Thisvector is then used to transfect cells which are simultaneously infectedwith vaccinia. Homologous recombination serves to insert the vacciniapromoter plus the gene encoding the instant protein into the viralgenome. The resulting TK⁻ recombinant can be selected by culturing thecells in the presence of 5-bromodeoxyuridine and picking viral plaquesresistant thereto.

An alternative route of administration involves gene therapy or nucleicacid immunization. Thus, nucleotide sequences (and accompanyingregulatory elements) encoding the subject GapC plasmin-binding proteinscan be administered directly to a subject for in vivo translationthereof. Alternatively, gene transfer can be accomplished bytransfecting the subject's cells or tissues ex vivo and reintroducingthe transformed material into the host. DNA can be directly introducedinto the host organism, i.e., by injection (see InternationalPublication No. WO/90/11092; and Wolff et al. (1990) Science247:1465-1468). Liposome-mediated gene transfer can also be accomplishedusing known methods. See, e.g., Hazinski et al. (1991) Am. J. Respir.Cell Mol. Biol. 4:206-209; Brigham et al. (1989) Am. J. Med. Sci.298:278-281; Canonico et al. (1991) Clin. Res. 39:219A; and Nabel et al.(1990) Science 249:1285-1288. Targeting agents, such as antibodiesdirected against surface antigens expressed on specific cell types, canbe covalently conjugated to the liposomal surface so that the nucleicacid can be delivered to specific tissues and cells susceptible toinfection.

Diagnostic Assays

As explained above, the GapC plasmin-binding proteins of the presentinvention may also be used as diagnostics to detect the presence ofreactive antibodies of streptococcus, for example S. dysgalactiae, in abiological sample in order to determine the presence of streptococcusinfection. For example, the presence of antibodies reactive with GapCplasmin-binding proteins can be detected using standard electrophoreticand immunodiagnostic techniques, including immunoassays such ascompetition, direct reaction, or sandwich type assays. Such assaysinclude, but are not limited to, Western blots; agglutination tests;enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidintype assays; radioimmunoassays; immunoelectrophoresis;immunoprecipitation, etc. The reactions generally include revealinglabels such as fluorescent, chemiluminescent, radioactive, enzymaticlabels or dye molecules, or other methods for detecting the formation ofa complex between the antigen and the antibody or antibodies reactedtherewith.

The aforementioned assays generally involve separation of unboundantibody in a liquid phase from a solid phase support to whichantigen-antibody complexes are bound. Solid supports which can be usedin the practice of the invention include substrates such asnitrocellulose (e.g., in membrane or microtiter well form);polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex(e.g., beads or microtiter plates); polyvinylidine fluoride; diazotizedpaper; nylon membranes; activated beads, magnetically responsive beads,and the like.

Typically, a solid support is first reacted with a solid phase component(e.g., one or more GapC plasmin-binding proteins) under suitable bindingconditions such that the component is sufficiently immobilized to thesupport. Sometimes, immobilization of the antigen to the support can beenhanced by first coupling the antigen to a protein with better bindingproperties. Suitable coupling proteins include, but are not limited to,macromolecules such as serum albumins including bovine serum albumin(BSA), keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art. Other molecules that can be used to bind the antigens to thesupport include polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and the like. Suchmolecules and methods of coupling these molecules to the antigens, arewell known to those of ordinary skill in the art. See, e.g., Brinkley,M. A. Bioconjugate Chem. (1992) 3:2-13; Hashida et al., J. Appl.Biochem. (1984) 6:56-63, and Anjaneyulu and Staros, International J. ofPeptide and Protein Res. (1987) 30:117-124.

After reacting the solid support with the solid phase component, anynon-immobilized solid-phase components are removed from the support bywashing, and the support-bound component is then contacted with abiological sample suspected of containing ligand moieties (e.g.,antibodies toward the immobilized antigens) under suitable bindingconditions. After washing to remove any non-bound ligand, a secondarybinder moiety is added under suitable binding conditions, wherein thesecondary binder is capable of associating selectively with the boundligand. The presence of the secondary binder can then be detected usingtechniques well known in the art.

More particularly, an ELISA method can be used, wherein the wells of amicrotiter plate are coated with a GapC plasmin-binding protein. Abiological sample containing or suspected of containing anti-GapCplasmin-binding protein immunoglobulin molecules is then added to thecoated wells. After a period of incubation sufficient to allow antibodybinding to the immobilized antigen, the plate(s) can be washed to removeunbound moieties and a detectably labeled secondary binding moleculeadded. The secondary binding molecule is allowed to react with anycaptured sample antibodies, the plate washed and the presence of thesecondary binding molecule detected using methods well known in the art.

Thus, in one particular embodiment, the presence of bound anti-GapCplasmin-binding antigen ligands from a biological sample can be readilydetected using a secondary binder comprising an antibody directedagainst the antibody ligands. A number of anti-bovine immunoglobulin(Ig) molecules are known in the art which can be readily conjugated to adetectable enzyme label, such as horseradish peroxidase, alkalinephosphatase or urease, using methods known to those of skill in the art.An appropriate enzyme substrate is then used to generate a detectablesignal. In other related embodiments, competitive-type ELISA techniquescan be practiced using methods known to those skilled in the art.

Assays can also be conducted in solution, such that the GapCplasmin-binding proteins and antibodies specific for those proteins formcomplexes under precipitating conditions. In one particular embodiment,GapC plasmin-binding proteins can be attached to a solid phase particle(e.g., an agarose bead or the like) using coupling techniques known inthe art, such as by direct chemical or indirect coupling. Theantigen-coated particle is then contacted under suitable bindingconditions with a biological sample suspected of containing antibodiesfor the GapC plasmin-binding proteins. Cross-linking between boundantibodies causes the formation of particle-antigen-antibody complexaggregates which can be precipitated and separated from the sample usingwashing and/or centrifugation. The reaction mixture can be analyzed todetermine the presence or absence of antibody-antigen complexes usingany of a number of standard methods, such as those immunodiagnosticmethods described above.

In yet a further embodiment, an immunoaffinity matrix can be provided,wherein a polyclonal population of antibodies from a biological samplesuspected of containing anti-GapC plasmin-binding molecules isimmobilized to a substrate. In this regard, an initial affinitypurification of the sample can be carried out using immobilizedantigens. The resultant sample preparation will thus only containanti-streptococcus moieties, avoiding potential nonspecific bindingproperties in the affinity support. A number of methods of immobilizingimmunoglobulins (either intact or in specific fragments) at high yieldand good retention of antigen binding activity are known in the art. Notbeing limited by any particular method, immobilized protein A or proteinG can be used to immobilize immunoglobulins.

Accordingly, once the immunoglobulin molecules have been immobilized toprovide an immunoaffinity matrix, labeled GapC plasmin-binding proteinsare contacted with the bound antibodies under suitable bindingconditions. After any non-specifically bound antigen has been washedfrom the immunoaffinity support, the presence of bound antigen can bedetermined by assaying for label using methods known in the art.

Additionally, antibodies raised to the GapC plasmin-binding proteins,rather than the GapC plasmin-binding proteins themselves, can be used inthe above-described assays in order to detect the presence of antibodiesto the proteins in a given sample. These assays are performedessentially as described above and are well known to those of skill inthe art.

The above-described assay reagents, including the GapC plasmin-bindingproteins, or antibodies thereto, can be provided in kits, with suitableinstructions and other necessary reagents, in order to conductimmunoassays as described above. The kit can also contain, depending onthe particular immunoassay used, suitable labels and other packagedreagents and materials (i.e. wash buffers and the like). Standardimmunoassays, such as those described above, can be conducted usingthese kits.

Deposits of Strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following strains wasmade with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va., under the provisions of the Budapest Treaty.The accession number indicated was assigned after successful viabilitytesting, and the requisite fees were paid. The designated deposits willbe maintained for a period of thirty (30) years from the date ofdeposit, or for five (5) years after the last request for the deposit,whichever is longer. Should a culture become nonviable or beinadvertently destroyed, or, in the case of plasmid-containing strains,lose its plasmid, it will be replaced with a viable culture(s) of thesame taxonomic description.

Should there be a discrepancy between the sequence presented in thepresent application and the sequence of the gene of interest in thedeposited plasmid due to routine sequencing errors, the sequence in thedeposited plasmid controls.

Bacterial ATCC Strain Plasmid Gene Deposit Date No. E. coli pET15bgapCgapC May 31, 2000 PTA-1976 BL21 DE3 (S. dysgalactiae) E. coli pMF521cgapC May 31, 2000 PTA-1975 BL21 DE3 (S. agalactiae) E. coli pMF521a gapCMay 31, 2000 PTA-1973 BL21 DE3 (S. uberis) E. coli pMF521e gapC May 31,2000 PTA-1972 BL21 DE3 (S. iniae)

3. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Materials and Methods

Enzymes were purchased from commercial sources, and used according tothe manufacturers' directions.

In the isolation of DNA fragments, except where noted, all DNAmanipulations were done according to standard procedures. See, Sambrooket al., supra. Restriction enzymes, T₄ DNA ligase, E. coli, DNApolymerase 1I, Klenow fragment, and other biological reagents can bepurchased from commercial suppliers and used according to themanufacturers' directions. Double stranded DNA fragments were separatedon agarose gels.

Sources for chemical reagents generally include Sigma Chemical Company,St. Louis, Mo.; Alrich, Milwaukee, Wis.; Roche Molecular Biochemicals,Indianapolis, Ind.

EXAMPLE 1 Preparation Amplification, Sequencing, Expression,Purification and Characterization of the S. dysgalactiae GapC PlasminBinding Protein

A. Preparation of S. dysgalactiae Chromosomal DNA

A clinical S. dysgalactiae isolate from a case of bovine mastitis (ATCCAccession No. ATCC43078) was obtained from the American Type CultureCollection (10801 University Boulevard, Manassas, Va. 20110-2209), andwas used as a source of DNA. The organism was routinely grown on TSAsheep blood agar plates (PML Microbiologicals, Mississauga, Ontario) at37° C. for 18 hours, or in Todd-Hewitt broth (Oxoid Ltd., Hampshire,England) supplemented with 0.3% yeast extract (THB-YE) at 37° C., 5%CO₂.

Chromosomal DNA was prepared from S. dysgalactiae grown in 100 ml ofTHB-YE supplemented with 20 mM glycine for approximately 6 hours, untilan A₆₀₀ of 0.8 to 1.0 was reached. Cells were harvested and re-suspendedin 50 mM EDTA, 50 mM Tris-HCl, 0.5% Tween-20® (Sigma, St. Louis, Mo.)and supplemented with RNase A (200 mg/ml), proteinase K (20 mg/ml),lysozyme (100 mg/ml) and mutanolysin (100 mg/ml). (SIGMA, St. Louis,Mo.). Following bacterial lysis for 30 minutes at 37° C. with vigorousshaking, guanidine hydrochloride and Tween-2®, pH 5.5, were mixed withthe lysate to give a final concentration of 0.8 M and 5%, respectively.This mixture was incubated at 50° C. for 30 minutes. The chromosomal DNAwas then purified using a Qiagen genomic-tip 100 g (Qiagen, SantaClarita, Calif.) and precipitated using 0.7 volumes of isopropanol. Theresulting pellet was washed in 70% ethanol and re-suspended in 0.5 ml 10mM Tris-HCl, pH 8.8.

B. Amplification and Cloning of the S. dysgalactiae gapC Gene

The gapC gene was amplified by PCR (See Mullis et al., U.S. Pat. No.4,683,195; Mullis, U.S. Pat. No. 4,683,202; ). The forward primer,gapC1, contained an Nde1 restriction (SEQ ID NO:1, shown in Table 1) andthe reverse primer, gapC1r, contained a BamHI site (SEQ ID NO:2, shownin Table 1). In the preceding primer sequences, depicted in Table 1,underlining denotes nucleotides added to the original sequence, andbolding indicates the location of restriction endonuclease recognitionsites.

PCR was carried out using Vent DNA polymerase (New England Biolabs,Mississauga, ON, Canada). 0.7 μg of S. dysgalactiae chromosomal DNA wasincubated in a reaction mixture containing 1 μM of each of the precedingprimers, 200 μM each of dATP, dTTP, dCTP and dGTP, 3 mM MgSO₄, 1×concentration of Thermopol buffer (New England Biolabs, Mississauga, ON,Canada) and 2 units Vent DNA polymerase. This mixture was incubated for3 amplification cycles of 1 minute at 94° C., 3 minutes at 50° C. and 1minute, 10 seconds at 72° C., then for 27 amplification cycles at 15seconds at 95° C., 30 seconds at 55° C., and 1 minute at 72° C., andfinally for 1 cycle of 5 min at 72° C.

TABLE 1 Sequence Identification Numbers and Corresponding Nucleotide andAmino Acid Sequences SEQ ID NO. Name Sequence 1 Primer gapC15′-GG CGG CGG  CAT  ATG GTA GTT AAA GTT GGT ATT AAC GG-3′ 2 PrimergapC1r 5′-GC  GGA   TCC  TTA TTT AGC GAT TTT TGC AAA GTA CTC-3′ 3Streptococcus dysgalactiae (see FIG. 1) gapC gene 4 Streptococcusdysgalactiae GapC protein 5 Streptococcus (see FIG. 2) agalactiae gapCgene 6 Streptococcus agalactiae GapC protein 7 Streptococcus uberis (seeFIG. 3) gapC gene 8 Streptococcus uberis GapC protein 9 Streptococcusparauberis (see FIG. 4) gapC gene 10 Streptococcus parauberis GapCprotein 11 Streptococcus iniae (see FIG. 5) gapC gene 12 Streptococcusiniae GapC protein

The gapC PCR product was cloned into the expression vector pET15B(Novagen, Madison, Wis.) which had been digested with BamHI and NdeI.Cloning of the PCR product into this site results in the addition of anin-frame coding sequence for a hexahistidyl tag to the gapC codingsequence. Subsequent expression yields a full-length protein with anattached histidine tag, which permits purification of the protein undernon-denaturing conditions using metal chelate chromatography.

This construct was used to transform E. coli BL21 DE3 (LifeTechnologies, Gaithersburg, Md.). This transformed strain was designatedBL21 DE3 (pET15bgapC).

C. Isolation of Chromosomal DNA and Amplification and Cloning of thegapC Gene from S. agalactiae. S. uberis, S. parauberis and S. iniae

The gapC gene were prepared from other isolates essentially as describedabove.

Chromosomal DNA from S. agalactiae, S. uberis, and S. parauberis wasisolated from strains obtained from the American Type Culture Collection(10801 University Boulevard, Manassas, Va. 20110-2209; strainsdesignated ATCC 27541, 9927, and 13386, respectively). Chromosomal DNAfrom S. iniae was isolated from a strain designated 9117 obtained fromMount Sinai Hospital, University of Toronto.

The primers used to amplify the gapC genes from the Streptococcusstrains listed above were the same as those used in the case of S.dysgalactiae, i.e., primer gapC1 (SEQ ID NO:1) and primer gapclr (SEQ IDNO:2).

After amplification, the PCR product in each case was cloned intopPCR-Script, using the cloning protocol described in the PCR-Script Ampcloning Kit (Stratagene, La Jolla, Calif.). The PCR product insert wasthen excised using NdeI and BamHI and re-cloned into those sites inpE15b using conventional cloning protocols (See e.g., Sambrook et al.,supra.). The plasmids containing the S. agalactiae, S. uberis, S.parauberis and S. iniae were designated pMF521c, pMF521a, pMF521d, andpMF521e, respectively.

D. Nucleotide Sequence of the gapC Gene and Deduced Amino Acid Sequences

Sequences homologous to gapC of S. equisimilis homolog (Gase, et al.(1996) European J. of Biochem. 239:42-51) were originally identifiedwhile sequencing a linked but unrelated gene of S. dysgalactiae. Toobtain the complete sequence of the S. dysgalactiae gapC gene, PCR wasemployed using the primers described above, i.e., primer gapC1 andprimer gapC1r.

The sequence was determined using fluorescence tag terminators on an ABI373 DNA automatic sequencer (Applied Biosystems, Emeryville, Calif.) atthe Plant Biotechnology Institute (PBI, Saskatoon, Canada).

both 1 depicts the coding sequence of the gapC gene from S. dysgalactiae(DysGapC) (SEQ ID NO:3) and the deduced amino acid sequence (SEQ IDNO:4).

The sequences of the GapC proteins isolated form S. agalactiae, S.uberis, S. parauberis, and S. iniae were determined by the same method.

FIGS. 2 through 5 depict both the nucleotide sequences and the predictedamino acid sequences for the S. dysgalactiae GapC protein (DysgalGapC)(SEQ ID NO:3 and SEQ ID NO:4), as well as for the GapC proteins of S.agalactiae (AgalGapC) (SEQ ID NO:5 and SEQ ID NO:6), S. uberis(UberGapC) (SEQ ID NO:7 and SEQ ID NO:8), S. parauberis (PUberGapC) (SEQID NO:9 and SEQ ID NO:10), and S. iniae (IniaeGapC) (SEQ ID NO:11 andSEQ ID NO:12), respectively.

The S. dysgalactiae GapC protein gene depicted in FIGS. 1A-1B codes fora 336 amino acid protein which does not appear to contain either asignal sequence or membrane anchor domain. A search of the GenBankdatabase using the BLASTX program revealed that the open reading framewas 95.5% homologous to GapC of S. equisimilis (GenBank Accession No.X97788) and 99.4% homologous to GapC of S. pyogenes (GenBank AccessionNo. M95569). The predicted amino acid sequence of the GapC protein alsoexhibited 43% amino acid identity to bovine glyceraldehyde-3-phosphatedehydrogenase (GenBank Accession No. U85042).

Similarly, for the S. agalactiae, S. uberis, S. parauberis and S. iniaeGapC protein sequences, neither signal sequences nor membrane anchordomains appear to be present.

Sequence homologies are tabulated in Table 2.

TABLE 2 Sequence Homologies Between Various GapC Protein Sequences S.equisimilis S. pyogenes Bovine GAPDH S. dysgalactiae  95.5%  94.4% 43%S. agalactiae 87.02% 91.07% (not determined) S. parauberis 86.31% 90.77%S. uberis 88.39% 92.26% S. iniae 86.31% 89.88%E. Expression and Purification of the Recombinant S. dysgalactiae GapCPlasmin Binding Protein

The Hexahistidyl-tagged GapC protein was expressed and purified undernon-denaturing conditions using metal chelate (Ni-NTA) affinitychromatography.

E. coli BL21 DE3 containing the recombinant plasmid was grown in LuriaBroth, containing 100 μg/ml ampicillin to an A₆₀₀ of approximately 0.5.Expression of the GapC protein was then induced by the addition of 1 mMisopropyl-β,D-thiogalactoside (IPTG) (Sigma, St. Louis, Mo.]. Followingthree hours incubation at 37° C., cells were harvested, washed in columnbuffer (50 mM sodium phosphate buffer, pH 8.0, 0.3 M NaCl, 10 mMimidazole) and lysed by sonication.

Approximately 40% of the recombinant protein was in the soluble fractionof the cell sonicate with a yield of approximately 50 mg of therecombinant protein per liter of culture volume, determined with a DCProtein Assay Kit (Bio-Rad Laboratories, Mississauga, ON, Canada) usingbovine serum albumin (Pierce, Rockford, Ill.) as a standard.

The lysate was cleared by centrifugation and the soluble fraction wasapplied to a Ni-NTA column (Qiagen), which was subsequently washed with10 column volumes of column buffer (as above, except containing 20 mMimidazole). The Hexahistidyl-tagged GapC was eluted using column buffer(as above, except containing 250 mM imidazole), yielding a homogenousprotein fraction having a GapC concentration of 10-15 mg/ml. Thatfraction was dialyzed against 2000 volumes of PBSA (136 mM sodiumchloride, 2.6 mM potassium chloride, 8.1 mM sodium phosphate dibasic,1.46 mM potassium phosphate monobasic).

F. Expression and Purification of Recombinant GapC Protein from S.agalactiae, S. uberis, S. parauberis, and S. iniae

Expression and purification of the recombinant proteins from thesestreptococcus species is accomplished by the same methods described inExample 1E, above. The transformed bacterial strains used to express theS. agalactiae, S. uberis, S. parauberis and S. iniae recombinant GapCproteins were designated BL21 DE3 (pMF521c), BL21 DE3 (pMF521a), BL21DE3 (pMF521d), and BL21 DE3 (pMF521e), respectively.

G. Characterization of the Recombinant S. dysgalactiae GapC Protein

1. SDS-Page Analysis

SDS-polyacrylamide gel electrophoresis was performed on a sample of theeluted protein using the method described by Laemli (Laemli, U. K.(1970) Nature 227:680-685). The results are presented in FIG. 18. In thefigure: lane 1, molecular weight markers (20.5 to 103 kDa range; BioRadLaboratories, Emeryville, Calif.); lane 2, soluble recombinant S.dysgalactiae GapC protein purified by Ni-NTA affinity chromatography.

These results demonstrate that purification by affinity chromatographyon a Ni-NTA column yielded a homogenous protein fraction.

2. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Activity ofRecombinant GapC and S. dysgalactiae Whole Cells

GAPDH catalyzes the oxidative phosphorylation ofD-glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate in the presenceof NAD⁺ and inorganic phosphate. The high degree of homology of GapC tostreptococcal glyceraldehyde-3-phosphate dehydrogenase suggested thatGapC may exhibit this enzyme activity.

The GAPDH activity of S. dysgalactiae whole cells (10¹⁰ CFU) and therecombinant GapC protein was determined by measuring the reduction ofNAD⁺ to NADH. The assay buffer was composed of 40 mM triethanolamine, 50mM Na₂ HPO₄ and 5 mM EDTA, pH 6.8. S. dysgalactiae cells or 5 mg ofpurified recombinant protein were incubated in assay buffer containing 7ml glyceraldehyde-3-phosphate (49 mg/ml; Sigma Chemical Company), 75microliters NAD⁺ (15 mM; Sigma Chemical Company) in a final volume of 1ml. Negative controls consisted of samples which did not containglyeraldehyde-3-phosphate or the recombinant GapC molecule/S.dysgalactiae cells. The reduction of NAD⁺ to NADH was monitoredspectrophotometrically at an Absorbance of 340 nanometers.

The results indicated that both the recombinant protein as well asintact wild-type S. dysgalactiea cells had enzymatic activity (notshown). Furthermore, when S. dysgalactiae cells were treated withTrypsin to digest surface proteins, GAPDH activity disappeared. Thus,the enzymatic activity observed for the intact wild type cells was notdue to intracellular GAPDH.

This data suggests that the GapC protein is localized on the cellsurface despite the apparent lack of either a signal sequence or amembrane anchoring region in either the nucleotide or amino acid.

3. Plasmin-Binding Activity of Recombinant S. dysgalactiae GapC PlasminBinding Protein and S. dysgalactiae whole cells.

A microplate assay was used to determine if the recombinant GapC proteinwas capable of binding bovine plasmin, and if so whether the boundplasmin was in an enzymatically active form.

Ninety-six-well microtiter plates were coated with 5 mg of purifiedrecombinant GapC protein, washed 3 times with 0.1% gelatin-PBSA with0.05% TWEEN-20 (PBSGT). The wells were blocked for one hour at 37° C. inthe same buffer, washed, and incubated with 200 ml of bovine plasmin(0.25 mg/ml; Boehringer Mannheim, Indianapolis, Ind.) for 1 hr at 37° C.The wells were then washed 8 times with PBSGT. 200 ml of the syntheticsubstrate chromazine-PL (Tos-Gly-Pro-Lys-4-NA, 0.3 mg/ml) were added tothe wells and incubated at 37° C. for one hour. The presence ofassociated plasmin activity was determined by measuring the level ofparanitroanalide (4-nitraniline) released into the supernatant anddetected based on an Absorbance of 405 nanometers. A similar procedurewas used to measure plasmin-binding activity of S. dysgalactiae wholecells, with the exception that 10¹⁰ cells were washed with PB SGT andre-suspended in 400 μl chromazine-PL (0.3 mg/ml) and incubated for 1hour at 37° C.

The results, shown in FIG. 19, demonstrate that the purified recombinantprotein was capable of binding enymatically active bovine plasmin.Likewise, when S. dysgalactiae whole cells were utilized, similarresults were obtained. In the figure, the data represents the mean ofthree individual assays.

Thus, the plasmin-receptor is located on the surface of S. dysgalactiaeand the purified protein retains biological activity.

EXAMPLE 2 Immunization with S. dysgalactiae GapC and ExperimentalInfection of Cattle

Vaccines were formulated in such a fashion that they contained 50 mg/mlof affinity purified recombinant GapC in the oil-based adjuvant VSA3(VIDO, Saskatoon, Saskatchewan, Canada). VSA3 is a combination ofEmulsigen Plus™ (MVP Laboratories, Ralston, Nebr.) andDimethyldioctadecyl ammonium bromide (Kodak, Rochester, N.Y.). Theaffinity-purified recombinant GapC protein used for the vaccinepreparation is shown in FIG. 18.

Twenty-four non-lactating Holsteins with no history of S. dysgalactiaeinfection were obtained from various farms in Saskatchewan, Canada. Oneweek prior to vaccination, all animals were treated with Cepha-dry™(Ayerst Laboratories, Montreal, Canada) (300 mg per quarter), in orderto clear any infection of the udders prior to the vaccination step.

Groups of 8 animals were immunized subcutaneously with two doses ofvaccines containing S. dysgalactiae GapC, Mig (an Fc receptor proteinisolated from S. dysgalactiae which was evaluated simultaneously), or aplacebo with a three-week interval between immunizations. Two weeksfollowing the second immunization, animals were exposed to 650 colonyforming units of S. dysgalactiae delivered into three quarters with anudder infusion cannula. The fourth quarter on each animal served as anun-infective control.

All animals were examined daily for clinical signs of disease andsamples from all udder quarters were collected on each day. Samples wereobserved for consistency and somatic cell counts as well as bacterialnumbers were determined.

EXAMPLE 3 Determination of GapC-Specific Antibodies

GapC-specific antibodies in bovine serum were measured using anenzyme-linked immunosorbent assay (ELISA). Briefly, microtiter plates(NUNC, Naperville, Ill.) were coated by adding 1 microgram per wellpurified recombinant antigen in 50 mM sodium carbonate buffer, pH 9.6,and incubated overnight at 4° C. The liquid was removed and the wellswere blocked with 3% bovine serum albumin for 1 hr at 37° C. Serialdilutions of bovine serum (from 1 in 4 to 1 in 6,400) were then added tothe wells and incubated for 2 hours at room temperature. The wells wereaspirated, washed and incubated with 100 ml of alkalinephosphatase-conjugated goat anti-bovine IgG (Kirkgaard & PerryLaboratories Inc., Gaithersburg, Md.) for 1 hr at room temperature. Thewells were washed again, and 100 μl of p-nitrophenol phosphate (SIGMA,St. Louis, Mo.) was added as a substrate to detect alkaline phosphataseactivity. The absorbance at 405 nanometers was recorded following 1 hrincubation with the substrate at room temperature.

Where referred to in the figures, the specific antibody titer isexpressed as the reciprocal of the dilution showing activity abovebackground levels.

EXAMPLE 4 Bacterial Colonization

Bacteria were enumerated by spreading serial dilutions (10⁰ to 10⁻³)directly onto TSA sheep blood agar plates followed by overnightincubation at 37° C., 5% CO₂. Colonization is defined as >500 cfu/ml ofthe challenge organism recovered.

To confirm that the bacteria recovered from milk secretions were S.dysgalactiae, selected colonies recovered from each animal were testedusing an API strep-20 test (bioMerieux SA, Hazelwood, Mo.) according tothe manufacturer's instructions. This test is a standardized methodcombining 20 biochemical tests for the determination of enzymaticactivity and fermentation of sugars. The reactions are read according toa reading table and the identification is obtained by either referringto the analytical profile index or using identification software.

Following challenge, animals from all groups were shown to be colonizedby S. dysgalactiae (FIG. 20). Only the GapC-immunized cows had astatistically significant reduction in the number of infected quartersand total numbers of bacteria isolated per quarter. Therefore,immunization with GapC reduced bacterial colonization followingchallenge with S. dysgalactiae.

The relationship between anti-GapC titer and bacterial colonization isshown in FIGS. 21 and 22. There was a strong correlation betweenanti-GapC serum antibody level and the maximum number of bacteria(expressed in CFU (log₁₀)/ml milk) found in any quarter r=0.74) (FIG.21) as well as the total number of infected quarters (r²=0.74) (FIG.22). Correlation was calculated using GraphPad Prism software, version2.01 (GraphPad Software Inc., San Diego, Calif.).

This correlation is also illustrated in FIGS. 23 and 24 where theGapC-immunized group is subdivided into high titer and low titerresponders. In these figures, “low titer responders” refer to the fouranimals with the poorest response against GapC while “high titerresponders” refer to the remainder of the group. No colonizationoccurred in the high titer group, while even the low titer group showedreduced numbers of bacteria recovered after day 3.

EXAMPLE 5 Determination of Inflammatory Response

Inflammatory response was measured as a function of somatic cell count(i.e., lymphocytes, neutrophils, and monocytes). Somatic cell countswere measured in a Coulter counter using standard techniques, asrecommended by Agriculture and Agri-Food Canada Pamphlet IDF50B (1985)Milk and Milkproducts—Methods of Sampling. Samples were always readwithin 48 hours of collection and fixation, at days 1 through 7 postchallenge.

The numbers of somatic cells present in the gland was determined on eachday post challenge. Numbers from the unchallenged quarter remainedconstant throughout the trial while on day 1, the GapC group was lowerthan the placebo-immunized group (FIG. 25). The difference between theGapC and the placebo groups was statistically significant. Theindividual data from day 1 is shown in FIG. 26; data for GapC-treatedanimals over a 7 day period post-challenge is shown in FIG. 27. Samplesfrom the quarters of GapC-immunized animals were indistinguishable fromunchallenged quarters.

Therefore, immunization with GapC reduced the inflammatory responsefollowing challenge with S. dysgalactiae.

EXAMPLE 6 Cross-Protection Against S. uberis Infection

During the last 3 days of the vaccine trial, a mixed population ofbacteria were recovered from the mammary gland secretions. Furtheranalysis with the API 20 Strep test confirmed the identity of thestrains present in the mixture as S. dysgalactiae and S. uberis, thelatter representing a natural infection which occurred during the trial.The group which was immunized with GapC appeared to be significantlycross-protected against S. uberis colonization (see FIG. 20), indicatingthat GapC may be a broadly cross-protective antigen capable ofprotecting against infection by multiple Streptococcal species.

Thus, vaccination with S. dysgalactiae GapC is therefore capable ofproviding cross-protection against S. uberis infection.

Thus, the cloning, expression and characterization of various GapCplasmin binding proteins is disclosed, as are methods of using the same.Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

1. An isolated Streptococcus dysgalactiae GapC protein comprising theamino acid sequence of SEQ ID NO:4.
 2. A vaccine composition comprisinga pharmaceutically acceptable vehicle and the GapC protein according toclaim
 1. 3. The vaccine composition of claim 2, further comprising anadjuvant.
 4. A method of producing a vaccine composition comprising thesteps of (1) providing the GapC protein according to claim 1, and (2)combining said GapC protein with a pharmaceutically acceptable vehicle.5. An immunodiagnostic test kit for dytecting Streptococcusdysagalaction infection, said test kit comprising the GapC proteinaccording to claim 1 and instructions for conducting theimmunodiagnostic test.